The transplant community has sought to achieve immune tolerance to allografts for decades, both for the health benefit of the patient and as a cost saving measure. In collaboration with ITN, we are conducting a tolerance trial using T-cell depletion in humans. This study would be augmented greatly by knowing whether immunosuppression (IS) can be withdrawn completely based on mechanistic assays. Our goal here is to use a preclinical non-human primate (NHP) renal transplant model as a means of validating 1) tolerance induction regimens and 2) clinically applicable immune assays to assist in determining whether patients can be safely withdrawn from IS. In this application, we hypothesize that the allo-specific memory response can be blocked sufficiently after depletion, and regulatory T cell activity enhanced, such that long term transplant tolerance in NHP renal transplant recipients will be achieved. Further, we hypothesize that the acquisition and loss of tolerance is measurable and predictable by the proposed assays. We base these hypotheses on the following observations. First, lymphocyte depletion alone in NHP or in humans does not generally result in tolerance induction but does increase the relative frequency of T cells with an effector (TEFF) or memory (TMEM) phenotype. Second, with the addition of short-term IS, T-cell depletion in NHPs has been shown to prolong allograft survival and to create donor-specific unresponsiveness. Third, regulatory T-cell (TREG) activity can be permitted, even enhanced, in vivo through the use of common IS drugs in rodent models. Lastly, we have developed an in vitro assay in humans, which when tested in rodents accurately reflects the immune status toward the graft. Specific Aim 1 is to characterize TMEM cell expansion in T-cell depleted NHP renal transplant recipients and the impact tacrolimus (TAC) on controlling the TMEM population. We will a) examine the effects of TAC with or without sirolimus (SRL) on TMEM cell recovery following anti-CD3 immunotoxin (IT)-mediated T-cell depletion in rhesus macaques receiving a life sustaining renal allograft, b) examine TMEM cell activity in vitro during T cell recovery, c) monitor factors that influence T cell depletion and recovery. For Specific Aim 2, we will transplant a second set of monkeys and treat with IT/steroid/TAC/SRL to test a novel set of immune tolerance assays. We will a) determine allograft tolerance status while recipient NHPs are on SRL monotherapy, b) assign monkeys to either tolerant or non-tolerant groups and subsequently withdraw SRL, c) test for tolerance with a donor skin graft, and d) monitor TREG cell frequency and activity. In Specific Aim 3 we will determine whether TREG cell activity inversely correlates with the progression of chronic allograft nephropathy (CAN). We will a) monitor changes in TREG activity with the loss of tolerance/progression of CAN, b) determine whether resumption of SRL treatment blocks or delays further loss of kidney function due to CAN.